Resumo: Angiotensina II, um peptídeo hipertensivo, e bradicinina, um peptídeo hipotensivo, são fatores humorais cruciais para a regulação da pressão sanguínea. A enzima chave desse sistema é a enzima conversora de angiotensina que produz angiotensina II a partir de angiotensina I e degrada bradicinina. A descoberta dos primeiros inibidores naturais dessa enzima, os peptídeos potenciadores de bradicinina (BPPs), tornou possível o desenvolvimento dos primeiros medicamentos utilizados no controle da pressão arterial humana. Caracteristicamente, os BPPs contêm de 5 a 13 resíduos de aminoácidos apresentando um resíduo de piroglutâmico no N-terminal e um resíduo de prolina no Cterminal. O precursor de BPPs encontrado na glândula de veneno de Bothrops jararaca contém 256 resíduos de aminoácidos e codifica para sete BPPs alinhados em tandem seguidos pelo peptídeo natriurético tipo-C. Até o momento, não se conhecem os mecanismos envolvidos para a liberação desses peptídeos da proteína precursora. Dessa forma, a resolução da estrutura dessa proteína pode contribuir para a elucidação do mecanismo evolvido no processamento do precursor para a liberação dos BPPs. Duas construções da proteína precursora de BPPs (domínio BPP e domínios BPP+CNP) da glândula de veneno de B. jararaca foram expressas, purificadas e suas identidades confirmadas por experimentos de western blotting. A pureza das amostras foi avaliada por SDS-PAGE e a presença de enovelamento após expressão heteróloga foi observada por experimentos de dicroísmo circular e fluorescência. Os ensaios de cristalização não foram promissores. Isso pode ser explicado pela baixa concentração da proteína usada no experimento. Assim, devido ao baixo nível de expressão de ambas as proteínas, métodos para maximização da expressão foram empregados resultando em significante...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Angiotensin II, a hypertensive peptide, and bradykinin, a hipotensive peptide, are crucial humoral factors for the regulation of blood pressure. The key enzyme for this system is the angiotensin-converting enzyme that produces angiotensin II from angiotensin I and degrades bradykinin. The discovery of the first natural inhibitors for this enzyme, the bradykinin potentiating peptides (BPPs), made it possible to develop the early drugs aimed at controlling unbalanced cardiovascular functions. Characteristically, BPPs contain 5 to 13 amino acid residues that have a pyroglutamyl residue at the N-terminus and a praline residue at the C-terminus. The BPP precursor protein contains 256 amino acid residues coding for seven BPPs aligned in tandem followed by the C-type natriuretic peptide. At present, there are no suggested mechanisms for understanding the release of BPPs from the precursor protein. Two constructs of the BPP precursor protein (BPP domain and BPP+CNP domains) from the venom gland of Bothrops jararaca were over-expressed, purified and the identity of both recombinant proteins confirmed by western blotting experiments. The purity of the samples was assessed by SDS-PAGE and the protein fold after expression was observed by circular dichroism and fluorescence experiments. Crystallization assays were not successful, probably due to the low protein concentration used for the experiment. Considering the low expression level observed for both recombinant proteins, the experimental methods were optimized to maximize the yield and resulted in high protein amounts in inclusion bodies. Methods were applied aiming at the solubilization of the proteins from the insoluble fraction and protein purification under denaturing conditions was carried out yielding high amounts of pure protein. Until this moment, none of the procedures were successful in producing refolded proteins. Work ...(Complete abstract click electronic access below) / Orientador: Raghuvir Krishnaswamy Arni / Coorientador: Mirian Akemi Furue Hayashi / Banca: Fátima Pereira de Souza / Banca: Patrick Jack Spencer / Mestre

Orientador: Raghuvir Krishnaswamy Arni / Banca: Adélia Cristina Oliveira Cintra / Banca: Patrick Jack Spencer / Abstract: Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out. / Mestre